Name: 'bioluminescent' reporter phage for the detection of category a bacterial pathogens
Uploaded: 2011-07-08T12:00:00.000+00:00
Duration: 11 min 31 s
Description: Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

Questa ricerca è stata sostenuta dal programma di ricerca Piccole Imprese per l&#39;Innovazione del National Institutes of Health (NIAID, 1R43AI082698-01) e l&#39;USDA Istituto Nazionale di Alimentazione e l&#39;Agricoltura (NIFA, 2009-33610-20028).

1. Y. pestis piastra inoculazione <ol><li> Streak Y. pestis A1122 (BeiResources # NR15) colture su Luria-Bertani (LB) agar (Miller). Utilizzare una tecnica sterile ed eseguire tutte le Y. manipolazioni pestis in un tipo di classe II A cabinet. biosicurezza Y. pestis A1122 è un livello di biosicurezza (BSL) 2 ceppo esclusi agente selezionare. Manca sia la coppia 75 kb basso contenuto di calcio di risposta (LCR) plasmide di virulenza, e il luogo pgm che sono necessari per la virulenza. Crescere Y. pestis a 28 ° C per 48 ore a temperatura statica aria incubatrice. Il tasso di crescita di Y. pestis è lenta con un tempo di generazione di 1,25 h 9. </li></ol> 2. Y. pestis liquido mezzi inoculazione <ol><li> Trasferire un singolo Y. colonia pestis in una sterile 17 x 100 mm Tubo di coltura contenente 2 ml di brodo LB sterile utilizzando un anello di metallo inoculazione. Seleziona una colonia che è uniforme nelle dimensioni, è indicativo delle altre colonie, ed è chiaramente separato dal resto delle colonie sulla piastra. Tubo vorticoso brevemente e far crescere la cultura a 28 ° C per circa 20 ore in una agitazione incubatore (225 giri). </li></ol> 3. Y. pestis escrescenza, fago Inoltre giornalista, e il rilevamento bioluminescenti <ol><li> Diluire la notte Y. pestis 1:20 cultura in brodo LB fresco in un tubo da 50 ml falco (ad esempio, 500 ml di cellule in 9,5 ml di terreno) e crescono a 28 ° C con agitazione (225 giri). Crescere fino a una densità ottica a 600 nm (A 600) di 0,2 si raggiunge (circa 5 h). Cellule in attiva crescita sono da preferire per il rilevamento in quanto la capacità del fago per trasdurre una risposta bioluminescente è correlato alla vitalità e benessere dei batteri ospitante. Tuttavia, il sistema di rilevazione ha dimostrato di essere compatibile con le cellule raccolte in tutte le fasi del ciclo di crescita 7. </li><li> Generare il substrato necessario per la reazione bioluminescente preparando un 2% di n-decanale soluzione. Mescolare 200 ml di n-decanale con 9,8 ml di brodo LB. Energicamente per 5 s. Primo la micropiastra luminometro iniettore con 2 mL di n-2% decanale soluzione. Pre-impostare il luminometro al autoinject 67 microlitri della soluzione del decanale ad ogni micropiastra bene e poi subito a leggere il campione per 10 s. </li><li> Dispensare 1 ml di brodo LB in 3 provette di coltura. Aggiungere 20 ml di soluzione madre giornalista fago (stock di 5 x 10 9 unità placca formando [UFP] / mL) a ciascuna provetta, il fago e dei media di campioni da solo funge da controllo negativo. In assenza di Y. cellule pestis, l&#39;aggiunta del fago giornalista non dovrebbe suscitare una reazione bioluminescente. </li><li> Dispensare 1 ml di Y. pestis cultura (dal punto 3.1) in 6 tubi cultura. Aggiungere 20 ml di stock fago giornalista a 3 delle culture (culture di prova). I restanti 3 culture servire come &#39;cellule solo&#39; di controllo negativo (autobioluminescence sfondo). Mescolare culture vortexando brevemente, e la raccolta 200 ul di ciascun campione (per leggere il tempo 0) ed erogare in un piatto bianco microtitolo a 96 pozzetti. Incubare la cultura rimanendo a 28 ° C con agitazione (225 giri). </li><li> Misurare il tempo 0 campioni per bioluminescenza (unità di luce relativa, RLU) utilizzando il luminometro per micropiastre. </li><li> Harvest 200 ul di ciascun campione dopo 10, 20, 30, 40, 50 e 60 minuti post-giornalista Inoltre fago, e misurare il campione per bioluminescenza. Se Y. pestis cellule sono presenti, il fago giornalista si legano specificamente alla cellula, inietta il suo DNA, e utilizzare i padroni di casa macchinario trascrizionale e traslazionale di trascrivere e tradurre i geni reporter luxAB (Figura 1). </li><li> La forza del segnale e il tempo di risposta del segnale è proporzionale al numero di cellule presenti. Ad alte concentrazioni di cellule (10 5 -10 8 UFC / ml), un significativo aumento RLU rispetto ai controlli dovrebbe essere evidente entro 20 min. A concentrazioni cella inferiore, (10 2 -10 4 UFC / ml), un significativo aumento RLU dovrebbe essere evidente entro 40-60 min. </li><li> In alternativa, per accelerare il processo di rilevamento senza la necessità di Y. escrescenza pestis, una colonia da un piatto fresco cresciuto possono essere mescolati (nel vortex) direttamente in una provetta Eppendorf 1,5 ml con 200 ml di brodo LB ospitare il fago reporter. Dispensare la cellula / fago miscela in un pozzo della micropiastra. Incubare la micropiastra a 28 ° C e leggere il campione per bioluminescenza dopo 60 min (punto di volta sola). </li></ol> 4. Rappresentante dei risultati: Un rappresentante esperimento durata di fago giornalista mediata rilevamento di Y. pestis è illustrato nella figura 2. I controlli negativi di 1) fago da solo (senza cellule), o 2) le cellule da solo (senza fago) forniscono livelli basali di bioluminescenza di circa 20 RLU tutto il min 60 incubation (livelli basali sono specifici luminometro). Al contrario, un aumento della bioluminescenza per i campioni di prova (fagi giornalista e cellule) è evidente a 15 minuti dopo l&#39;aggiunta dei fagi. La potenza del segnale dovrebbe aumentare costantemente più di 60 min. Incubazioni per periodi di tempo prolungati (oltre 80 min), si tradurrà in un segnale che scenderà dal segnale di picco a causa di fagi lisi mediata delle cellule ospiti. Risultati simili si ottengono a temperature di incubazione di 37 ° C anche se la temperatura ottimale per Y. crescita pestis è di 28 ° C 9. <img src="/files/ftp_upload/2740/2740fig2.jpg" alt="Figura 2"/> Figura 2. Fago-mediata rilevazione bioluminescenti di Y. pestis. Al tempo 0, fagi giornalista e le cellule sono state mescolate, incubate a 28 ° C, e la bioluminescenza (RLU) è stata monitorata nel tempo. Un aumento significativo RLU (* Studenti t-test, p &lt;0,05) è evidente entro 15 min.

<ol>
	<li>Darling, R. G., Catlett, C. L., Huebner, K. D., Jarrett, D. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Threats+in+bioterrorism.+I:+CDC+category+A+agents.">Threats in bioterrorism. I: CDC category A agents.</a> Emerg Med Clin North Am. 20, 273-309 (2002).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Laboratory manual of plague diagnostic tests.. , (2000).</li><li>. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> , (1999).</li><li>Inglesby, T. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Anthrax+as+a+biological+weapon,+2002:+updated+recommendations+for+management.">Anthrax as a biological weapon, 2002: updated recommendations for management.</a> JAMA. 287, 2236-2252 (2002).</li><li>Schuch, R., Fischetti, V. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+genomic+analysis+of+the+Wbeta+and+gamma+phages+infecting+Bacillus+anthracis:+implications+for+evolution+of+environmental+fitness+and+antibiotic+resistance.">Detailed genomic analysis of the Wbeta and gamma phages infecting Bacillus anthracis: implications for evolution of environmental fitness and antibiotic resistance.</a> J Bacteriol. 188, 3037-3051 (2006).</li><li>Garcia, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genome+sequence+of+Yersinia+pestis+bacteriophage+phiA1122+reveals+an+intimate+history+with+the+coliphage+T3+and+T7+genomes.">The genome sequence of Yersinia pestis bacteriophage phiA1122 reveals an intimate history with the coliphage T3 and T7 genomes.</a> J Bacteriol. 185, 5248-5262 (2003).</li><li>Schofield, D. A., Molineux, I. J., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnostic+bioluminescent+phage+for+detection+of+Yersinia+pestis.">Diagnostic bioluminescent phage for detection of Yersinia pestis.</a> Journal of Clinical Microbiology. 47, 3887-3894 (2009).</li><li>Schofield, D. A., Westwater, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Phage-mediated+bioluminescent+detection+of+Bacillus+anthracis.">Phage-mediated bioluminescent detection of Bacillus anthracis.</a> Journal of Applied Microbiology. 107, 468-478 (2009).</li><li>Chu, M. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> CDC: Basic laboratory protocols for the presumptive identification of Yersinia pestis. , 1-19 (2001).</li><li>Gunnison, J. B., Larson, A., Lazarus, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+differentiation+between+Pasteurella+pestis+and+Pasteurella+pseudotuberculosis+by+action+of+bacteriophage.">Rapid differentiation between Pasteurella pestis and Pasteurella pseudotuberculosis by action of bacteriophage.</a> J Infect Dis. 88, 254-255 (1951).</li><li>Lazarus, A. S., Gunnison, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Action+of+Pasteurella+pestis+Bacteriophage+on+Strains+of+Pasteurella,+Salmonella,+and+Shigella.">The Action of Pasteurella pestis Bacteriophage on Strains of Pasteurella, Salmonella, and Shigella.</a> J Bacteriol. 53, 705-714 (1947).</li><li>Sergueev, K. V., He, Y., Borschel, R. H., Nikolich, M. P., Filippov, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+sensitive+detection+of+Yersinia+pestis+using+amplification+of+plague+diagnostic+bacteriophages+monitored+by+real-time+PCR.">Rapid and sensitive detection of Yersinia pestis using amplification of plague diagnostic bacteriophages monitored by real-time PCR.</a> PLoS One. 5, e11337-e11337 (2010).</li></ol>

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Questo metodo dimostra la capacità del fago giornalista per rilevare rapidamente Y. pestis dal fago giornalista può trasdurre una risposta bioluminescente segnale colto Y. pestis cellule entro 20 minuti dopo l&#39;aggiunta dei fagi. Il fago giornalista è anche in grado di rilevare direttamente Y. pestis in matrici clinica, senza il presupposto di isolamento e 7 successiva coltivazione. Rispetto ai test standard di lisi fagica che in genere richiedono 48 ore per il completamento, questo riduce sensibilmente i tempi di rilevazione. Studi precedenti hanno dimostrato che il wild-type fago ΦA1122 può lisare quasi tutti naturali Y. pestis isolati, e &#39;specifico&#39; per Y. pestis 6,10,11, tuttavia, alcuni Y. ceppi pseudotuberculosis hanno dimostrato di essere ΦA1122 suscettibili, se coltivate a temperature superiori a 20 ° C 6,10,12. La ragione per la temperatura-sensibili suscettibilità differenziale è sconosciuta, ma presumibilmente a causa della temperatura-dipendente cambiamenti negli strati superficiali della cellula / composizione. Quindi, un avvertimento potenziale del sistema di rilevazione dei fagi giornalista è la possibilità di un falso positivo di risposta con ceppi dal strettamente legati specie Y. pseudotuberculosis. Esecuzione del test dei fagi giornalista alla temperatura restrittiva (20 ° C) impedisce un falso segnale positivo in campioni che possono contenere Y. pseudotuberculosis. Così, la specificità può essere strettamente controllati durante l&#39;utilizzo culture isolate cresciute ad una temperatura specifica. In sintesi, rapida individuazione e la diagnosi di Y. pestis è essenziale per una prognosi positiva in quanto la peste, la peste polmonare in particolare, è quasi sempre fatale se il trattamento non viene somministrato entro le prime 24 ore dopo l&#39;insorgenza dei sintomi. Questo metodo ha il potenziale per soddisfare queste esigenze per l&#39;identificazione degli isolati confermato coltivate o all&#39;interno di matrici di rilevanza clinica.

<table border="1"><tbody><tr><th> Nome del reagente </th><th> Azienda </th><th> Numero di catalogo </th></tr><tr><td> Difco agar LB, Miller </td><td> VWR </td><td> 90003-346 </td></tr><tr><td> Difco LB brodo, Miller </td><td> VWR </td><td> 90003-350 </td></tr><tr><td> 17 x 100 mm cultura tubi </td><td> USA scientifico </td><td> 1485-0810 </td></tr><tr><td> n-decanale </td><td> Sigma </td><td> D7384 </td></tr><tr><td> Veritas luminometro per micropiastre </td><td> Turner Biosystems </td><td> 9100-001 </td></tr><tr><td> Microlite micropiastra a 96 pozzetti </td><td> VWR </td><td> 62402-984 </td></tr></tbody></table>

'bioluminescent' reporter phage for the detection of category a bacterial pathogens

Yersinia pestis e Bacillus anthracis sono di categoria A batteri patogeni che sono gli agenti causali della peste e antrace, rispettivamente 1. Anche se la presenza naturale di entrambe le malattie &#39;è ora relativamente rara, la possibilità di gruppi terroristici con questi patogeni come arma biologica è reale. A causa della intrinseca comunicabilità della malattia, rapido decorso clinico, e alto tasso di mortalità, è fondamentale che un focolaio essere rilevati rapidamente. Pertanto le metodologie che consentono il rilevamento rapido e la diagnosi sono essenziali per garantire l&#39;attuazione immediata di misure di sanità pubblica e l&#39;attivazione della gestione delle crisi. Fago ricombinante giornalista può fornire un approccio rapido e specifico per l&#39;individuazione di Y. pestis e B. anthracis. I Centri per il Controllo delle Malattie e la Prevenzione attualmente utilizzano il classico test lisi dei fagi per l&#39;identificazione ha confermato di questi batteri patogeni 2-4. Questi test sfruttare naturale fago che sono specifici e litico per i loro ospiti batterici. Dopo una crescita durante la notte del batterio coltivato in presenza del fago specifico, la formazione di placche (lisi batterica) fornisce una identificazione positiva del target batterico. Anche se questi test sono robusti, soffrono da tre difetti: 1) sono di laboratorio, 2) che richiedono l&#39;isolamento e la coltura batterica dal campione sospetto, e 3) prendono 24-36 ore per essere completato. Per affrontare questi problemi, ricombinante &quot;light-tag&quot; fago giornalista sono stati geneticamente modificati, integrando le Vibrio harveyi luxAB geni nel genoma di Y. pestis e B. anthracis fago specifico 5-8. La risultante dei fagi giornalista luxAB sono stati in grado di rilevare i loro target specifico, mediante una rapida (pochi minuti) e sensibile che conferisce un fenotipo bioluminescente di cellule riceventi. È importante sottolineare che il rilevamento è stato ottenuto sia con cellule riceventi coltivate o con campioni clinici mock-infettate 7. A scopo dimostrativo, qui si descrive il metodo per il fago-mediata rilevamento di un noto Y. pestis isolare con un fago luxAB giornalista costruito dalla peste CDC diagnostica fago ΦA1122 6,7 (Figura 1). Un metodo simile, con piccole modifiche (ad esempio cambiamento della temperatura di crescita e media), possono essere utilizzati per la rilevazione di B. anthracis isola con il B. anthracis fago Wβ reporter:: luxAB 8. Il metodo descrive il fago-mediata trasduzione di un fenotipo biolumescent ad essere coltivato Y. pestis cellule che vengono successivamente misurata con un luminometro per micropiastre. I principali vantaggi di questo metodo il test tradizionale lisi dei fagi è la facilità di utilizzo, i risultati rapidi e la possibilità di testare campioni multipli simultaneamente in un formato a 96 pozzetti di micropiastre. <img src="/files/ftp_upload/2740/2740fig1.jpg" alt="Figura 1"/> Figura 1. Schema di rilevamento. I fagi sono mescolati con il campione, il fago infetta il cellulare, luxAB sono espressi, e il bioluminesces cellula. Trattamento dei campioni non è necessaria; il fago e le cellule sono mescolate e successivamente valutate per la luce.

Watch this Scientific Journal Video about 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens at JoVE.com

'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Un metodo semplice per l&#39;identificazione di batteri patogeni priorità è quella di utilizzare fago giornalista geneticamente modificato. Queste fago reporter, che sono specifici per la loro specie ospite particolare, sono in grado di trasduzione rapidamente una risposta bioluminescente segnale alle cellule ospite. Qui, descriviamo l&#39;uso del fago giornalista per la rilevazione di<em&gt; Yersinia pestis</em&gt;.

Phage &#39;Bioluminescent&#39; Reporter per il rilevamento di batteri patogeni di categoria A

Yersinia pestis and Bacillus anthracis are Category A bacterial pathogens that are the causative agents of the plague and anthrax, respectively 1. ...

bioluminescent-reporter-phage-for-detection-category-bacterial

Universidad San Sebastian

Research

JoVE Journal

Immunology and Infection

13.4K Views.  Guild Associates, Inc.. Un metodo semplice per l'identificazione di batteri patogeni priorità è quella di utilizzare fago giornalista geneticamente modificato. Queste fago reporter, che sono specifici per la loro specie ospite particolare, sono in grado di trasduzione rapidamente una risposta bioluminescente segnale alle cellule ospite. Qui, descriviamo l'uso del fago giornalista per la rilevazione di Yersinia pestis.

Video: 'Bioluminescent' Reporter Phage for the Detection of Category A Bacterial Pathogens

Using Luciferase to Image Bacterial Infections in Mice

Imaging is a valuable technique that can be used to monitor biological processes. In particular, the presence of cancer cells, stem cells, specific immune cell types, viral pathogens, parasites and bacteria can be followed in real-time within living animals 1-2. Application of bioluminescence imaging to the study of pathogens has advantages as compared to conventional strategies for analysis of infections in animal models3-4. Infections can be visualized within individual animals over time, without requiring euthanasia to determine the location and quantity of the pathogen. Optical imaging allows comprehensive examination of all tissues and organs, rather than sampling of sites previously known to be infected. In addition, the accuracy of inoculation into specific tissues can be directly determined prior to carrying forward animals that were unsuccessfully inoculated throughout the entire experiment. Variability between animals can be controlled for, since imaging allows each animal to be followed individually. Imaging has the potential to greatly reduce animal numbers needed because of the ability to obtain data from numerous time points without having to sample tissues to determine pathogen load3-4.
This protocol describes methods to visualize infections in live animals using bioluminescence imaging for recombinant strains of bacteria expressing luciferase. The click beetle (CBRLuc) and firefly luciferases (FFluc) utilize luciferin as a substrate5-6. The light produced by both CBRluc and FFluc has a broad wavelength from 500 nm to 700 nm, making these luciferases excellent reporters for the optical imaging in living animal models7-9. This is primarily because wavelengths of light greater than 600 nm are required to avoid absorption by hemoglobin and, thus, travel through mammalian tissue efficiently. Luciferase is genetically introduced into the bacteria to produce light signal10. Mice are pulmonary inoculated with bioluminescent bacteria intratracheally to allow monitoring of infections in real time. After luciferin injection, images are acquired using the IVIS Imaging System. During imaging, mice are anesthetized with isoflurane using an XGI-8 Gas Anethesia System. Images can be analyzed to localize and quantify the signal source, which represents the bacterial infection site(s) and number, respectively. After imaging, CFU determination is carried out on homogenized tissue to confirm the presence of bacteria. Several doses of bacteria are used to correlate bacterial numbers with luminescence. Imaging can be applied to study of pathogenesis and evaluation of the efficacy of antibacterial compounds and vaccines.

Methods for bioluminescence imaging of bacterial infections in living animals are decribed. Pathogens are modified to express luciferase allowing optical whole body imaging of infections in live animals. Animal models can be infected with luciferase expressing pathogens and the resulting course of disease visualized in real-time by bioluminescence imaging.

Utilizzando luciferasi di infezioni batteriche immagine in topi

Imaging is a valuable technique that can be used to monitor biological processes.  In particular, the presence of cancer cells, stem cells, specific ...

Ricerca

Immunologia e infezioni

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite this similarity, these bacteria show extreme variations in characteristics like growth behavior, intensity of light emission or regulation of bioluminescence. The method presented here is a newly developed assay that combines recording of cell growth and bioluminescent light emission intensity over time utilizing a plate reader. The resulting growth and light emission characteristics can be linked to important features of the respective bacterial strain, such as quorum sensing regulation. The cultivation of a range of bioluminescent bacteria requires a specific medium (e.g., artificial sea water medium) and defined temperatures. The easy to handle, non-bioluminescent standard-research bacterium Escherichia coli (E. coli), on the other hand, can be cultivated inexpensively in high quantities in laboratory scale. Exploiting E. coli by introducing a plasmid containing the whole lux operon can simplify experimental conditions and additionally opens up many possibilities for future applications. The expression of all lux genes utilizing an E. coli expression strain was achieved by construction of an expression plasmid via Gibson cloning and insertion of four fragments containing seven lux genes and three rib genes of the lux-rib operon into a pET28a vector. E. coli based lux gene expression can be induced and controlled via Isopropyl-&#946;-D-thiogalactopyranosid (IPTG) addition resulting in bioluminescent E. coli cells. The advantages of this system are to avoid quorum sensing regulation restrictions and complex medium compositions along with non-standard growth conditions, such as defined temperatures. This system enables analysis of lux genes and their interplay, by the exclusion of the respective gene from the lux operon, or even addition of novel genes, exchanging the luxAB genes from one bacterial strain by another, or analyzing protein complexes, such as luxCDE.

In Situ Measurement and Correlation of Cell Density and Light Emission of Bioluminescent Bacteria

Bioluminescent bacteria regulate light production through a variety of mechanisms, such as quorum sensing. This novel method allows the in situ investigation of bioluminescence and the correlation of light emission to cell density. An artificial bioluminescent Escherichia coli system allows the characterization of lux operons, Lux proteins, and their interplay.

In Situ Misurazione e la correlazione di densità delle cellule ed emissione di luce di batteri bioluminescenti

There is a considerable number of bacterial species capable of emitting light. All of them share the same gene cluster, namely the lux operon. Despite ...

Biochimica

Biosensor for Detection of Antibiotic Resistant Staphylococcus Bacteria

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) antibody conjugated latex beads have been utilized to create a biosensor designed for discrimination of methicillin resistant (MRSA) and sensitive (MSSA) S. aureus species 1,2. The lytic phages have been converted into phage spheroids by contact with water-chloroform interface. Phage spheroid monolayers have been moved onto a biosensor surface by Langmuir-Blodgett (LB) technique 3. The created biosensors have been examined by a quartz crystal microbalance with dissipation tracking (QCM-D) to evaluate bacteria-phage interactions. Bacteria-spheroid interactions led to reduced resonance frequency and a rise in dissipation energy for both MRSA and MSSA strains. After the bacterial binding, these sensors have been further exposed to the penicillin-binding protein antibody latex beads. Sensors analyzed with MRSA responded to PBP 2a antibody beads; although sensors inspected with MSSA gave no response. This experimental distinction determines an unambiguous discrimination between methicillin resistant and sensitive S. aureus strains. Equally bound and unbound bacteriophages suppress bacterial growth on surfaces and in water suspensions. Once lytic phages are changed into spheroids, they retain their strong lytic activity and show high bacterial capture capability. The phage and phage spheroids can be utilized for testing and sterilization of antibiotic resistant microorganisms. Other applications may include use in bacteriophage therapy and antimicrobial surfaces.

Lytic phage biosensors and antibody beads are able to discriminate between methicillin resistant (MRSA) and sensitive staphylococcus bacteria. The phages were immobilized by a Langmuir-Blodgett method onto a surface of a quartz crystal microbalance sensor and worked as broad range staphylococcus probes. Antibody beads recognize MRSA.

Biosensori per il rilevamento di antibiotici batteri di stafilococco resistenti

A structurally transformed lytic bacteriophage having a broad host range of Staphylococcus aureus strains and a penicillin-binding protein (PBP 2a) ...

Bioingegneria

A Fluorescence-based Method to Study Bacterial Gene Regulation in Infected Tissues

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some factors act directly on virulence genes; others control pathogenesis by adjusting the expression of downstream regulators or the accumulation of signals that affect regulator activity. While regulation has been studied extensively during in vitro growth, relatively little is known about how gene expression is adjusted during infection. Such information is important when a particular gene product is a candidate for therapeutic intervention. Transcriptional approaches like quantitative, real-time RT-PCR and RNA-Seq are powerful ways to examine gene expression on a global level but suffer from many technical challenges including low abundance of bacterial RNA compared to host RNA, and sample degradation by RNases. Evaluating regulation using fluorescent reporters is relatively easy and can be multiplexed with fluorescent proteins with unique spectral properties. The method allows for single-cell, spatiotemporal analysis of gene expression in tissues that exhibit complex three-dimensional architecture and physiochemical gradients that affect bacterial regulatory networks. Such information is lost when data are averaged over the bulk population. Herein, we describe a method for quantifying gene expression in bacterial pathogens in situ. The method is based on simple tissue processing and direct observation of fluorescence from reporter proteins. We demonstrate the utility of this system by examining the expression of Staphylococcus aureus thermonuclease (nuc), whose gene product is required for immune evasion and full virulence ex vivo and in vivo. We show that nuc-gfp is strongly expressed in renal abscesses and reveal heterogeneous gene expression due in part to apparent spatial regulation of nuc promoter activity in abscesses fully engaged with the immune response. The method can be applied to any bacterium with a manipulatable genetic system and any infection model, providing valuable information for preclinical studies and drug development.

Described here is a method for analyzing bacterial gene expression in animal tissues at a cellular level. This method provides a resource for studying the phenotypic diversity occurring within a bacterial population in response to the tissue environment during an infection.

Un metodo basato su fluorescenza per studiare la regolazione genica batterica nei tessuti infetti

Bacterial virulence genes are often regulated at the transcriptional level by multiple factors that respond to different environmental signals. Some ...

Genetica

Detection of Bacteria Using Fluorogenic DNAzymes

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses each and every year. Prevention of such outbreaks and minimization of the impact of an ongoing epidemic place an ever-increasing demand for analytical methods that can accurately identify culprit pathogens at the earliest stage. Although there is a large array of effective methods for pathogen detection, none of them can satisfy all the following five premier requirements embodied for an ideal detection method: high specificity (detecting only the bacterium of interest), high sensitivity (capable of detecting as low as a single live bacterial cell), short time-to-results (minutes to hours), great operational simplicity (no need for lengthy sampling procedures and the use of specialized equipment), and cost effectiveness. For example, classical microbiological methods are highly specific but require a long time (days to weeks) to acquire a definitive result.1 PCR- and antibody-based techniques offer shorter waiting times (hours to days), but they require the use of expensive reagents and/or sophisticated equipment.2-4 Consequently, there is still a great demand for scientific research towards developing innovative bacterial detection methods that offer improved characteristics in one or more of the aforementioned requirements. Our laboratory is interested in examining the potential of DNAzymes as a novel class of molecular probes for biosensing applications including bacterial detection.5
DNAzymes (also known as deoxyribozymes or DNA enzymes) are man-made single-stranded DNA molecules with the capability of catalyzing chemical reactions.6-8 These molecules can be isolated from a vast random-sequence DNA pool (which contains as many as 1016 individual sequences) by a process known as "in vitro selection" or "SELEX" (systematic evolution of ligands by exponential enrichment).9-16 These special DNA molecules have been widely examined in recent years as molecular tools for biosensing applications.6-8
Our laboratory has established in vitro selection procedures for isolating RNA-cleaving fluorescent DNAzymes (RFDs; Fig. 1) and investigated the use of RFDs as analytical tools.17-29 RFDs catalyze the cleavage of a DNA-RNA chimeric substrate at a single ribonucleotide junction (R) that is flanked by a fluorophore (F) and a quencher (Q). The close proximity of F and Q renders the uncleaved substrate minimal fluorescence. However, the cleavage event leads to the separation of F and Q, which is accompanied by significant increase of fluorescence intensity.
More recently, we developed a method of isolating RFDs for bacterial detection.5 These special RFDs were isolated to "light up" in the presence of the crude extracellular mixture (CEM) left behind by a specific type of bacteria in their environment or in the media they are cultured (Fig. 1). The use of crude mixture circumvents the tedious process of purifying and identifying a suitable target from the microbe of interest for biosensor development (which could take months or years to complete). The use of extracellular targets means the assaying procedure is simple because there is no need for steps to obtain intracellular targets.
Using the above approach, we derived an RFD that cleaves its substrate (FS1; Fig. 2A) only in the presence of the CEM produced by E. coli (CEM-EC).5 This E. coli-sensing RFD, named RFD-EC1 (Fig. 2A), was found to be strictly responsive to CEM-EC but nonresponsive to CEMs from a host of other bacteria (Fig. 3).
Here we present the key experimental procedures for setting up E. coli detection assays using RFD-EC1 and representative results.

We have recently reported a novel approach for generating fluorogenic DNAzyme probes that can be applied to set up a simple, "mix-and-read" fluorescent assay for bacterial detection. These special DNA probes catalyze the cleavage of a chromophore-modified DNA-RNA chimeric substrate in the presence of crude extracellular mixture (CEM) produced by a specific bacterium, thereby translating bacterial detection into fluorescence signal generation. In this report we will describe key experimental procedures where a specific DNAzyme probe denoted "RFD-EC1" is employed for the detection of the model bacterium, Escherichia coli (E. coli).

Rilevazione di batteri utilizzando DNAzimi fluorogenici

Outbreaks linked to food-borne and hospital-acquired pathogens account for millions of deaths and hospitalizations as well as colossal economic losses ...

Bioluminescent Bacterial Imaging In Vivo

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of bacteria in gene and cell therapy for cancer. Bacteria present an attractive class of vector for cancer therapy, possessing a natural ability to grow preferentially within tumors following systemic administration. Bacteria engineered to express the lux gene cassette permit BLI detection of the bacteria and concurrently tumor sites. The location and levels of bacteria within tumors over time can be readily examined, visualized in two or three dimensions. The method is applicable to a wide range of bacterial species and tumor xenograft types. This article describes the protocol for analysis of bioluminescent bacteria within subcutaneous tumor bearing mice. Visualization of commensal bacteria in the Gastrointestinal tract (GIT) by BLI is also described. This powerful, and cheap, real-time imaging strategy represents an ideal method for the study of bacteria in vivo in the context of cancer research, in particular gene therapy, and infectious disease. This video outlines the procedure for studying lux-tagged E. coli in live mice, demonstrating the spatial and temporal readout achievable utilizing BLI with the IVIS system.

Bioluminescent Bacterial Imaging In Vivo

This article describes the administration of lux-tagged bacteria to mice and subsequent in vivo analysis using IVIS bioluminescence imaging.

Bioluminescent Imaging batterica<em&gt; In Vivo</em

This video describes the use of whole body bioluminesce imaging (BLI) for the study of bacterial trafficking in live mice, with an emphasis on the use of ...

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast enhancement in vitro and in vivo. FUEL shares many of the same underlying principles as Bioluminescence Resonance Energy Transfer (BRET), yet greatly differs in the acceptable working distances between the luminescent source and the fluorescent entity. While BRET is effectively limited to a maximum of 2 times the F&ouml;rster radius, commonly less than 14 nm, FUEL can occur at distances up to &micro;m or even cm in the absence of an optical absorber. Here we expand upon the foundation and applicability of FUEL by reviewing the relevant principles behind the phenomenon and demonstrate its compatibility with a wide variety of fluorophores and fluorescent nanoparticles. Further, the utility of antibody-targeted FUEL is explored. The examples shown here provide evidence that FUEL can be utilized for applications where BRET is not possible, filling the spatial void that exists between BRET and traditional whole animal imaging.

A Step Beyond BRET: Fluorescence by Unbound Excitation from Luminescence FUEL

Expanding the foundation and applicability of Fluorescence by Unbound Excitation from Luminescence (FUEL) by surveying the relevant principles and demonstrating its compatibility with a multitude of fluorophores and antibody-targeted conditions.

A Step Beyond BRET: Fluorescenza da Unbound eccitazione di luminescenza (FUEL)

Fluorescence by Unbound Excitation from Luminescence (FUEL) is a radiative excitation-emission process that produces increased signal and contrast ...

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome (HUS), colonize to the intestinal tract of humans. To study the detailed mechanism of EHEC colonization in vivo, it is essential to have animal models to monitor and quantify EHEC colonization. We demonstrate here a mouse-EHEC colonization model by transforming the bioluminescent expressing plasmid to EHEC to monitor and quantify EHEC colonization in living hosts. Animals inoculated with bioluminescence-labeled EHEC show intense bioluminescent signals in mice by detection with a non-invasive in vivo imaging system. After 1 and 2 days post infection, bioluminescent signals could still be detected in infected animals, which suggests that EHEC colonize in hosts for at least 2 days. We also demonstrate that these bioluminescent EHEC locate to mouse intestine, specifically in the cecum and colon, from ex vivo images. This mouse-EHEC colonization model may serve as a tool to advance the current knowledge of the EHEC colonization mechanism.

Detection of Enterohemorrhagic Escherichia Coli Colonization in Murine Host by Non-invasive In Vivo Bioluminescence System

A detailed protocol of a mouse model for enterohemorrhagic E. coli (EHEC) colonization by using bioluminescence-labeled bacteria is presented. The detection of these bioluminescent bacteria by a non-invasive in vivo imaging system in live animals can advance our current understanding of EHEC colonization.

Rilevamento della colonizzazione di Escherichia Coli enteroemorragica in murino Host di sistema Non invasivo In Vivo bioluminescenza

Enterohemorrhagic E. coli (EHEC) O157:H7, which is a foodborne pathogen that causesdiarrhea, hemorrhagic colitis (HS), and hemolytic uremic syndrome ...

Using Coculture to Detect Chemically Mediated Interspecies Interactions

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by secreting metabolites. These metabolites have the potential to modulate the physiology and differentiation of their microbial neighbors and are likely important factors in the establishment and maintenance of complex microbial communities. We have developed a fluorescence-based coculture screen to identify such chemically mediated microbial interactions. The screen involves combining a fluorescent transcriptional reporter strain with environmental microbes on solid media and allowing the colonies to grow in coculture. The fluorescent transcriptional reporter is designed so that the chosen bacterial strain fluoresces when it is expressing a particular phenotype of interest (i.e. biofilm formation, sporulation, virulence factor production, etc.) Screening is performed under growth conditions where this phenotype is not expressed (and therefore the reporter strain is typically nonfluorescent). When an environmental microbe secretes a metabolite that activates this phenotype, it diffuses through the agar and activates the fluorescent reporter construct. This allows the inducing-metabolite-producing microbe to be detected: they are the nonfluorescent colonies most proximal to the fluorescent colonies. Thus, this screen allows the identification of environmental microbes that produce diffusible metabolites that activate a particular physiological response in a reporter strain. This publication discusses how to: a) select appropriate coculture screening conditions, b) prepare the reporter and environmental microbes for screening, c) perform the coculture screen, d) isolate putative inducing organisms, and e) confirm their activity in a secondary screen. We developed this method to screen for soil organisms that activate biofilm matrix-production in Bacillus subtilis; however, we also discuss considerations for applying this approach to other genetically tractable bacteria.

Bacteria produce secreted compounds that have the potential to affect the physiology of their microbial neighbors. Here we describe a coculture screen that allows detection of such chemically mediated interspecies interactions by mixing soil microbes with fluorescent transcriptional reporter strains of Bacillus subtilis on solid media.

Utilizzo di co-coltura per rilevare chimicamente mediata interspecie Interazioni

In nature, bacteria rarely exist in isolation; they are instead surrounded by a diverse array of other microorganisms that alter the local environment by ...

Biologia

Tracciamento di patogeni di tutto l'organo tramite quantificazione con reporter fluorescente

High&#45;Resolution Three&#45;Dimensional Whole&#45;Organ Tomography of Microbial Infections

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen cells within this diverse environment significantly affects their stress levels, responses, fate, and contribution to the overall progression of the disease and treatment failure. However, due to the technical difficulties in locating &#181;m-sized pathogen cells within cm-sized host organs, this area of research has been relatively unexplored. Here, we present a method for addressing this challenge. We employ serial two-photon tomography and AI-enhanced image analysis to locate individual Salmonella cells throughout the entire spleen, liver lobes, and whole lymph nodes of infected mice. Using fluorescent reporters and in vivo antibody administration, the replication rate of single Salmonella cells, their local interaction with specific immune cells, and bacterial responses to antibiotics can be determined. These methodologies open avenues for a comprehensive examination of infections, their prevention, and treatment within the three-dimensional tissue context.

Autore in primo piano: Svelare le risposte batteriche agli antibiotici e al sistema immunitario nei tessuti

Here, we describe a procedure that enables organ-wide detection of pathogenic bacteria during infection and quantification of fluorescent reporter activities.

Tomografia tridimensionale tridimensionale ad alta risoluzione di infezioni microbiche

Most infections take place within three-dimensional host tissues with intricate anatomy and locally varying host physiology. The positioning of pathogen ...

Phage 'Bioluminescent' Reporter per il rilevamento di batteri patogeni di categoria A

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